We are investigating how heparan sulfate influences FGFR2b signaling in specific progenitor cell types in the epithelium. The exquisite control of growth factor function by HS is dictated by the tremendous structural heterogeneity of its sulfated modifications. It is not known how specific HS structures control growth factor-dependent progenitor expansion during organogenesis. We used bioengineered 3-O-sulfated-HS to investigate HS function. Defining the minimum saccharide sequences of HS that determine the selectivity and specificity of their function will facilitate the synthesis of HS mimetics to specifically increase progenitor expansion in vitro. The aim of expanding salivary progenitors in vivo will be useful for tissue engineering. Using autologous adult biopsy cells, which can be expanded and/or directed in vitro, is a proposed treatment to repair damaged organs. One such strategy includes in vivo transplantation of organoids or stem cells grown in spheroid culture. While organoid and sphere formation mimics some aspects of development, they do not fully recapitulate organogenesis as the complex information provided by the surrounding niche, such as mesenchyme, blood vessels and nerves are not present. This is a major problem for the generation of complex branched organs in which coordinated branching morphogenesis of multiple cell types in the fetal microenvironment drive organ development. We are using multiple factors including heparin sulfate mimics identified from studies of the organ-specific fetal microenvironment, to maintain and expand multiple adult epithelial salivary progenitors in salisphere culture. Moreover, physical recombination of the adult salispheres in a laminin extracellular matrix with salivary mesenchyme, containing endothelial and neuronal cells, induced branching morphogenesis. Markers of progenitors were maintained and developmental differentiation programs were initiated. Thus, organ development provides a template for adult organ regeneration, and delineation of secreted and physical cues from the fetal niche will be useful to develop novel regenerative therapies that instruct adult salispheres to resume a developmental program and to repair and regenerate branching organs. This project also involves generating knockout mice of the Hs3st isoforms to create a genetic toolkit to understand the basic biology of how 3-O-sulfation influences organ development and homeostasis. While we focus on the salivary gland, the mouse phenotypes direct us to investigate earlier stages of embryo development and to compare other organs that are affected. These mice are important tools to analyze 3-O-sulfation in salivary gland progenitors and to better understand the fine tuning of cellular responses to FGFRs and HS modifications.